Homocysteine levels correlate with velocimetric parameters in patients with erectile dysfunction undergoing penile duplex ultrasound

Abstract Introduction Hyperhomocysteinemia may contribute to the development of endothelial dysfunction and, consequently, atherosclerosis, a systemic disease involving the vessels that may affect the cavernous arteries leading to vasculogenic erectile dysfunction. Our study aims therefore to explore the relationship between homocysteine levels and velocimetric parameters detected by basal penile duplex ultrasound such as peak systolic velocity and flaccid penile acceleration in patients with erectile dysfunction. Methods A cross‐sectional study was conducted collecting clinical, metabolic, hormonal, and instrumental (basal penile duplex ultrasound) data in patients affected by vasculogenic erectile dysfunction. Results Data of 126 subjects affected by erectile dysfunction were collected. Mean age was 52.1 ± 12.6 years, whereas mean body mass index was 25.6 ± 4.0 kg/m2. Basal penile duplex ultrasound showed peak systolic velocity values of 13.1 ± 2.9 cm/s and mean flaccid penile acceleration of 2.28 ± 0.70 m/s2, with a strong correlation among these two parameters (r = 0.690; p < 0.001). Frankly pathological values of peak systolic velocity and flaccid penile acceleration were detected in 39.7% and 4.8% of the subjects examined, respectively. Mean homocysteine levels were 14.9 ± 9.5 μmol/l. Homocysteine values >15 μmol/l were found in 26% of the subjects with erectile dysfunction. Peak systolic velocity values and homocysteine levels showed an inverse correlation (r = –0.213; p = 0.03). Similarly, flaccid penile acceleration values were inversely correlated to homocysteine levels (r = –0.199; p = 0.05). In addition, an inverse correlation was found between both peak systolic velocity and flaccid penile acceleration and body mass index, atherogenic lipid pattern, and age. Homocysteine and metabolic parameters showed no significant correlations. Conclusion Hyperhomocysteinemia is highly prevalent in erectile dysfunction patients. The results of our study show that homocysteine levels correlate with velocimetric parameters assessed by basal penile duplex ultrasound, confirming the role of hyperhomocysteinemia in the genesis of erectile dysfunction of arterial origin.


INTRODUCTION
Erectile dysfunction (ED), defined as the persistent inability to obtain or maintain penile erection sufficient for a satisfactory sexual performance, 1 is a common condition in middle aged men. According to data from the Massachusetts Male Aging Study (MMAS), its prevalence increases with aging, affecting up to half of men aged 40-70 years and reaching 70% in older men. 2 The etiology of ED is multifactorial, involving vasculogenic, neurogenic, hormonal, and pharmacological causes, but impaired penile blood flow is the leading mechanism. 1 Normal erectile function depends on the release of nitric oxide (NO) in the penile tissue by the endothelium and neurons following the stimulation of non-adrenergic, non-cholinergic nerves. In the corpora cavernosa, NO binds to soluble guanylin cyclase, stimulating the production of 3′-5′-cyclic guanosine monophosphate (cGMP). The latter activates protein kinase G (PKG) to form a complex cGMP/PKG which induces the relaxation of smooth muscle with consequent hyperflow of arterial blood and the onset of penile tumescence. 3 One of the main mechanisms underlying vascular ED is the endothelial dysfunction, which is accompanied by impaired expression and activation of NO synthase (NOS). 4 Endothelial dysfunction represents the first step in the origin of atherosclerosis 5 and underlies the development of cardiovascular disease (CVD), explaining, at least in part, the close link between the latter and ED. Indeed, about the 20% of ED patients may have asymptomatic CVD and the extent of the latter strongly correlates with the severity of ED. 2 Recently, clinical and experimental findings have shown that hyperhomocysteinemia (HHcys) can alter the homeostasis of the endothelium and contribute to the development of atherosclerosis. 6 Homocysteine (Hcys) is a sulfur-containing amino acid deriving from excess dietary methionine though a process called "trans-sulfuration." On the opposite, Hcys can be re-methylated to methionine by the enzymes methionine-synthase and 5,10-methylenetetrahydofolate reductase (MTHFR), with vitamin B12 or folic acid (FA) as a cofactor, respectively. When the remethylation pathway of Hcys is defective, because of congenital (e.g., genetic defects of MTHFR) or acquired conditions (including vitamins B6 and B12 and FA deficiencies and renal impairment), HHcys may occur. 7 To explain the relationship between CVD and HHcys, three different pathophysiological mechanisms have been proposed: (1) the reduction of the bioavailability of NO at endothelial level, (2) the direct cytotoxic effect of Hcys mediated by oxidative stress, and (3) dysregulation of the folate metabolism. 8 According to a recent meta-analysis, HHcys is more often observed in subjects with ED rather than controls, with a standardized mean difference of 1.00 (95% confidence interval (CI) 0.65-1.35, p < 0.0001). 9 Moreover, MTHFR polymorphisms with lower enzyme activity (namely C677TT and A1298C) are associated to a threefold increased risk for ED. 10 Dynamic penile duplex ultrasound (D-PDU) classically represents the gold standard for the diagnostics of men with vascular ED. 11 After intracavernous injection of a vasoactive drug (e.g., 10 μg alprostadil), color doppler recording of both the cavernous arteries is performed: a peak systolic velocity (PSV) of 35 cm/s or greater indicates arterial sufficiency, whereas a PSV < 25 cm/s is considered diagnostic of arterial insufficiency. 12 In the last 20 years, duplex ultrasound evaluation of cavernosal PSV in the penile flaccid state has emerged as an alternative, faster, cheaper, and safer technique. 13 Indeed, a basal-PSV <13 cm/s is predictive for dynamic-PSV <25 and <35 cm/s with an accuracy of 89% and 82%, respectively, 14 without the possible side effects of intracavernous injection of the vasoactive drugs. In addition, the evaluation of flaccid penile acceleration (FPA), calculated using PSV, end diastolic velocity (EDV), and systolic rise time (SRT) according to the formula (PSV -EDV)/SRT, allows to identify subjects with high risk for CVD, because an FPA <1.17 m/s 2 is associated to a threefold increase of major adverse cardiovascular events. 15 Thus, the present study aims to evaluate the correlation between basal penile duplex ultrasound (B-PDU) data as objective indexes of endothelial function, cardiovascular risk factors, and circulating levels of homocysteine Hcys in patients affected by ED.

Study design and participants
We enrolled 126 consecutive andrological outpatients affected by ED from the Endocrinology Clinic, Clinic and Molecular Sciences Department, University Hospital "Ospedali Riuniti" of Ancona, Italy, from April 2015 to January 2020. Subjects were included according to the following criteria: any sexual activity in the last 6 months; International Index of Erectile Dysfunction (IIEF-5) ≤21; ability to understand and provide informed consent. The exclusion criteria were hormonal diseases or uncontrolled diabetes mellitus, recent cardiovascular or cerebrovascular accidents, hormone therapy, and assumption of antineoplastic drugs or of any drug with well-known negative effects on erectile function (e.g., β-blockers, antidepressants).
All participants provided written informed consent to take part to the study. The study protocol was approved by the local ethics committee (number of protocol 2017-0222 OR).

Outcome measures
All patients underwent full physical examination, including blood pressure, weight, and height measurement. The following biochemical parameters were considered: follicle-stimulating hormone  Note: Data are expressed as mean ± SD or N (%). Abbreviations: FPA, flaccid penile acceleration; PSV, peak systolic velocity.

RESULTS
Clinical and laboratory data of the included patients are listed in Note: Data are expressed as mean ± SD. Abbreviations: FPA, flaccid penile acceleration; PSV, peak systolic velocity. Figure 1A,B). Post hoc analysis showed 86.7% and 80% statistical power for PSV and FPA, respectively. Moreover, an inverse correlation was present between Hcys and PSV (r = -0.213; p = 0.03) and between Hcys and FPA (r = -0.199; p = 0.05) (Figure 2A,B). In addition, a signifi-cant inverse correlation was present between PSV and age (r = -0.484;   Table 6 and was not significant.

DISCUSSION
Emerging evidence suggests a significant association between HHcys and ED. In a recent study based on a wide population of 1318 subjects in which serum levels of Hcys, FA, and vitamin B12 were evaluated, Chen et al. 19 found out that HHcys is associated to a threefold reducing its bioavailability. 23 In addition, Hcys increases endothelin-1 expression, which acts as a potent vasoconstrictor. 5 Taken together, these actions result in the inability of the endothelium to regulate vascular tone. Lastly, HHcys causes an increase in production and release of reactive oxygen species (ROS), a family of molecules which damages the integrity of the endothelium favoring the development of atherosclerosis. 5 Anyway, besides the indirect effect on atherosclerosis, HHcys can also impair erectile function with several direct mechanisms. Indeed, HHcys is able to inhibit acetylcholine-induced relaxation and cGMP production in the rabbit corpus cavernosum in vitro, 23 whereas, in vivo, it decreases the local production of NO in cavernous sinus and vessels affecting the number and latency of erections after apomorphine injection. 24 In addition, a decrease of smooth muscle cells and an increase in the fibrosis degree in the corpora cavernosa has been recently observed in a rat model of HHcys. 25 From a clinical point of view, the association between HHcys and ED has been reported by several authors. 9,19,26,27 Interestingly, Sansone et al. 9 reported a higher frequency of HHcys in ED subjects without diabetes mellitus rather than diabetic subjects with ED. Even if it could seem counter-intuitive, it has been suggested that in the present of significant comorbidities such as diabetes, HHcys could act in a synergistic way and smaller changes in serum Hcys could become clinically relevant. In our study, a significant correlation between Hcys and B-PDU parameters was present. Moreover, the latter showed a significant correlation with several metabolic parameters such as BMI, fasting glycemia, LDL-and HDL-cholesterol, and triglycerides.
Interestingly, Hcys levels did not correlate with any other metabolic parameter, but a significant correlation with age emerged. HHcys is common among elderly people and it is often because of a low intake of FA and B vitamins. 28 Since FA acts as a cofactor in the re-methylation process of Hcys, 29  Taken together, these data suggest that patients with ED and HHcys could benefit from normalization of Hcys levels, but further studies are needed to confirm these findings.
The strength of our study is that we used for the first time the parameters of B-PDU, an inexpensive, non-invasive, and easyto-perform examination, to study the impact of HHcys on male sexual function. To the best of our knowledge, no other author has yet reported similar data on such a numerous group of subjects. Given the role of HHcys in the development of ED and, above all, their relationship with CVD, it seems essential to associate laboratory assessment with instrumental examination in identifying subjects at high cardiovascular risk. On the other hand, we are aware of the limitations of our work first. The cross-sectional nature of our study and the lack of an intervention to reduce Hcys levels requires further confirmation, ideally by randomized controlled trials. In addition, the assessment of FA levels and the MTHFR genotyping could be useful to identify the source of HHcys and to investigate whether the effectiveness of Hcyslowering interventions may be affected by this.

CONCLUSION
A significant association between hyperhomocysteinemia and velocimetric parameters assessed by basal penile duplex ultrasound was detected in the present study. Other metabolic parameters significantly correlated with both peak systolic velocity and flaccid penile acceleration, but not with homocysteine. Patients with arteriogenic erectile dysfunction showed higher homocysteine levels than patients with peak systolic velocity >13 cm/s. Hyperhomocysteinemia could therefore represent an independent risk factor for erectile dysfunction and, consequently, cardiovascular disease, but further studies are needed to investigate if normalization of homocysteine levels could improve erectile function and prevent forthcoming cardiovascular events.

ACKNOWLEDGMENT
Open Access Funding provided by Universita Politecnica delle Marche within the CRUI-CARE Agreement.

CONFLICT OF INTEREST
All authors declare that they have no conflict of interests.

FUNDING INFORMATION
This study did not receive funds.

AUTHOR CONTRIBUTIONS
Giancarlo Balercia designed the work and provided overall supervision of the project. Gianmaria Salvio, Melissa Cutini, Lara Giovannini, Michele Perrone, and Alessandro Ciarloni conducted medical visits.